Substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide ... containing sensory neuropeptides such as substance P (SP) and calcitonin ...
Sensory nerves in the interface membrane of aseptic loose hip prostheses M. Ahmed, J. Bergstr¨om, H. Lundblad, W. J. Gillespie, A. Kreicbergs From the Karolinska Institute, Stockholm, Sweden
e studied the presence of sensory nerves by immunohistochemistry in the interface membranes of hip prostheses after aseptic loosening. Substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) were analysed as was protein gene product (PGP) 9.5, a general marker for nerve fibres. We identified nerve fibres in all samples but differences in their density were found. SP- and NKA-positive fibres were predominantly non-vascular, forming varicose nerve terminals. CGRP-immunoreactive nerve fibres with varicose terminals were seen mostly close to blood vessels, but also as free nerve endings. Sensory neuropeptides participate not only in nociception but also stimulate immune cells to release cytokines. The presence of sensory nerves in the interface membrane may reflect a pathophysiological response contributing to the aseptic loosening of hip prostheses.
W
J Bone Joint Surg [Br] 1998;80-B:151-5. Received 9 July 1997; Accepted after revision 21 August 1997
Aseptic loosening of hip prostheses is a common complica1 tion which is often associated with pain and periprosthetic 2 bone resorption. The interface membrane shows chronic inflammatory changes with infiltration of lymphocytes, macrophages, fibroblasts and the formation of foreign-body 3-5 giant cells. It has been suggested that implant wear
M. Ahmed, MD, PhD, Registrar, Senior Research Fellow J. Bergstr¨om, MD, Senior Registrar H. Lundblad, MD, Registrar A. Kreicbergs, MD, PhD, Professor Department of Orthopaedics, Karolinska Hospital, S-171 76 Stockholm, Sweden. W. J. Gillespie, ChM, FRCS Ed, FRACS, Professor Clinical Research Unit, Princess Margaret Rose Orthopaedic Hospital, Fairmilehead, Edinburgh EH10 7ED, UK. Correspondence should be sent to Dr M. Ahmed. ©1998 British Editorial Society of Bone and Joint Surgery 0301-620X/98/18138 $2.00 VOL. 80-B, NO. 1, JANUARY 1998
particles initiate this chronic granulomatous inflamma5-7 tion. Biochemical and immunohistochemical studies have shown increased levels of prostaglandin E2, col6-10 lagenase and cytokines in the membrane. These cellular 11 mediators are strongly implicated in inflammation, bone 12 resorption and subsequent aseptic loosening of joint implants. We have previously reported the presence of nerve fibres containing sensory neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) in bone and 13,14 joint tissues. Sensory neuropeptides participate not 15-17 18,19 only in nociception and inflammation but have also 20 been implicated in immune regulation, and have been 21,22 shown to be involved in local bone metabolism. Studies have focused mainly on the role of cellular mediators in aseptic prosthetic loosening and none has analysed the presence of sensory neuropeptides in the interface membrane.
Materials and Methods We obtained nine interface membranes surrounding cemented femoral components from patients undergoing revision hip arthroplasty. There were four women and five men with a mean age of 70 years (60 to 86). The mean time to revision was 8 years (5 to 12). The primary hip arthroplasty (Charnley, Exeter or Howse) had been performed for osteoarthritis in seven patients and for rheumatoid arthritis in two. All had pain in the hip or thigh with radiological evidence of loosening at the time of revision. Preoperative cultures from the hip were all negative. The membranes were rinsed in 0.01 mol/l of phosphatebuffered saline (PBS) at pH 7.3 and fixed with 4% paraformaldehyde in 0.2 mol/l of S¨orensen phosphate buffer containing 0.2% picric acid at pH 7.3 for two days. They were soaked for at least two days in 20% sucrose in 0.1 mol/l of S¨orensen phosphate buffer containing sodium azide and bacitracin at pH 7.2 (Sigma Chemicals, St Louis, Missouri). Two samples of each specimen were included in the study. Four serial 15 m sections were analysed from each membrane specimen for each peptide. The frozen sections were mounted directly on SuperFrost/Plus glass slides and immunostained according to the avidin-biotin complex 151
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M. AHMED,
Table I.
¨ J. BERGSTROM,
H. LUNDBLAD,
W. J. GILLESPIE,
A. KREICBERGS
Characteristics of the primary antisera used in the study
Antigen
Nature
Raised in
Dilution
Absorption*
SP NKA CGRP PGP 9.5
Sensory neuropeptide Sensory neuropeptide Sensory neuropeptide General neuronal marker
Rabbit Rabbit Rabbit Rabbit
1:20 000 1:5000 1:20 000 1:10 000
50 l 50 l 50 l N/A
* antiserum absorbed with corresponding synthetic peptide; concentration which completely blocked the immunoreactivity Table II. Density of PGP 9.5-, CGRP-, SP- and NKAimmunoreactive fibres in the interface membrane from nine patients (see text) Case
PGP 9.5
CGRP
SP
NKA
1 2 3 4 5 6 7 8 9
+++ +++ ++ ++ +++ ++ +++ ++ ++
+++ ++ + + ++ + ++ + +
++ ++ + + ++ + + + +
++ ++ + + + + + + +
method. Briefly, the sections were rinsed for 5 2 minutes in PBS and incubated overnight in a humid atmosphere at 4°C with antiserum to substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP) (all Peninsula Laboratories Europe Ltd, St Helens, UK) and protein gene product 9.5 (PGP 9.5; UltraClone, Cambridge, UK). The characteristics of the primary antibodies used are shown in Table I. The sections were then rinsed in PBS (5 2 min) and incubated with biotinylated goat anti-rabbit antibodies (1:250; Vector Laboratories, Burlingame, California) for 30 minutes at room temperature. Finally, fluorescein isothiocyanate (FITC)-conjugated avidin (1:500; Vector Laboratories, Burlingame, California) was used to visualise the immunoreaction. Control staining was performed by omitting the primary antiserum. Addition of 50 l of the peptides (SP, NKA and CGRP) to the corresponding antiserum before application on tissue sections served as another control. A Nikon epifluorescence microscope (Eclipse E800; Nikon Corporation, Instruments Division, Yokohama, Japan) was used to analyse the sections. T-Max black-and-white film (Kodak, Rochester, Minnesota) was used for photography. We used a scale of one plus (+) to three plus (+++) for the semiquantitative assessment of nerve fibres identified in each tissue section; + indicated 1 to 3 nerve fibres per section in a 10 magnification field, ++ 4 to 6, and +++ 6 and more nerve fibres. We performed histological analysis of the interface membrane to assess inflammatory changes and to confirm the presence of blood vessels.
Results We identified nerve fibres immunoreactive to SP, NKA, CGRP and PGP 9.5 in the interface membranes from all
Fig. 1 Photomicrograph of the interface membrane, showing infiltration of chronic inflammatory cells such as macrophages and foreign-body giant cells (black arrows). The membrane is mainly composed of fibrovascular tissue (haematoxylin and eosin 125).
patients. There were clear differences in the density of nerve fibres in different areas of the sample and also in different individuals (Table II). Histological examination showed that the interface membrane was mainly composed of fibrovascular tissue with abundant fibroblasts, macro4,5 phages and foreign-body giant cells (Fig. 1). Serial sections of the interface membranes were analysed by immunohistochemistry and haematoxylin and eosin staining to determine the relationship of the nerve fibres to blood vessels. The occurrence of nerve fibres in the interface membrane was established by positive staining with the general neuronal marker PGP 9.5 (Fig. 2A) in serial sections. The sensory nerve fibres were not distributed uniformly. CGRP-positive fibres with varicose terminals were more numerous and were seen to be close to blood vessels. Immunoreactivity to CGRP was found in the nerve bundles and in single fibres (Fig. 2B). The single nerve fibres were arranged as free nerve endings between the cells. Serial sections of the membrane showed co-localisation of CGRP and PGP 9.5 in most of the nerve fibres. SP and NKA-positive fibres were predominantly nonvascular and were present as thin fibres and formed varicose nerve terminals (Fig. 2C). They had an almost identical distribution and serial sections showed coexistence of SP and NKA as well as SP and CGRP fibres in most of the nerve fibres (Figs 2B and 2C).
Discussion Our study has shown that the interface membrane in aseptic loose cemented hip prostheses is supplied by sensory nerve fibres immunoreactive to substance P, NKA and CGRP. These are synthesised in neurones of the dorsal root ganglia. SP and NKA belong to the tachykinin family and are THE JOURNAL OF BONE AND JOINT SURGERY
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Fig. 2 Immunofluorescence micrographs of sections of the interface membrane after incubation with antisera to PGP 9.5 (A), CGRP (B) and SP (C). PGP 9.5- and CGRP-immunoreactive nerve fibres (arrows) are observed mainly around the walls of blood vessels (v). Arrowheads in B and C show the presence of CGRP and SP immunoreactivities in the same nerve fibres in serial sections of the interface membrane, indicating their coexistence (v = wall of the blood vessel) (58 (A), 117 (B,C)).
15-17
important modulators of nociceptive signals. They are mainly present in small-sized neurones (type B) of the dorsal root ganglia and their projections are unmyelinated C- and thinly myelinated A-type fibres, commonly known 23-25 By contrast, a substantial proporas nociceptive fibres. tion of CGRP occurs in large-sized (type A) neurones from 26,27 which myelinated fibres arise. CGRP, often co-local26 ised with SP in unmyelinated C fibres, facilitates the release of SP at the level of the spinal cord and delays SP degradation, thereby potentiating the nociceptive 28-30 effects. Patients with loose hip prostheses often have pain of varying intensity and distribution. The presence of different types of nerve fibre immunoreactive to SP, NKA and CGRP and also the difference in nerve density in the interface membrane may explain the different pain sensations and intensities. It is known that nociceptors respond to 31 mechanical, thermal and chemical stimuli. In the interface membrane, increased levels of prostaglandin E2, one of the biochemical agents known to stimulate nociceptors, have been reported. The activation of nociceptive pathways by prostaglandins may be one of the many mechanisms involved in the transmission of pain from hips with loose prostheses. The term neurogenic inflammation was coined when it was found that antidromic stimulation of primary afferent fibres elicited vasodilatation, increased capillary permeability, extravasation of plasma proteins and subsequently local 18,19 Substance P and NKA have been shown to oedema. VOL. 80-B, NO. 1, JANUARY 1998
induce inflammatory signs along with the release of hista32 mine from most cells. Recently, activated mast cells have been identified in the interface membrane of aseptic loose 33 hip prostheses. The inflammation induced by tachykinins is known to be inhibited by neurokinin receptor antago34 19,35,36 nists and also by pretreatment with capsaicin, which specifically depletes sensory neuropeptides. CGRP has been shown to enhance SP-induced oedema in the 37 periphery. In patients with rheumatoid arthritis, and in rat adjuvent arthritis, increased expression of sensory neuropeptides has been reported in the synovial membrane and 38-41 synovial fluid. Sensory neuropeptides have also been 42,43 implicated in vasodilatation. In our study, CGRP-positive fibres were found mostly around the blood vessels. It may therefore be possible that sensory neuropeptides participate in the inflammatory events in the interface membrane. Recently, it has been shown that implant wear particles 44,45 induce local and systemic immune responses. Increased expression of interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-) has been reported in the macrophages and fibro6-10 blasts in the membrane and also in pelvic lymph nodes 22 in patients with hip arthroplasties. These cytokines have been implicated in inflammation and periprosthetic bone resorption. A number of studies have suggested a functional link between the nervous and immune systems. Sensory nerve fibres have been demonstrated in lymphoid organs 46,47 Fibrowith a synapse-like contact to immune cells.
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blasts, lymphocytes and macrophages are equipped with SP and NKA receptors and SP has been shown to induce the 48,49 release of IL-1, IL-6 and TNF- from these cells. SP has also been reported to promote inflammatory cell chem50 51 otaxis, fibroblast proliferation and antibody forma52 tion. Furthermore, it has been shown that SP enhances the secretion of prostaglandin E2 and collagenase in synovio53 cytes and that CGRP stimulates lymphocyte prolifera54 tion. It is therefore possible that sensory neuropeptides may stimulate the different types of immune cells in a paracrine fashion to promote release of various proinflammatory mediators. Moreover, SP has recently been shown to induce bone resorption both in vitro and in 22 vivo. This study was supported by grants from the Swedish Medical Research Council (12X-08652-01), The Royal 80 Year Fund of King Gustaf V, Uggla’s Foundation and Karolinska Institute Research Funds. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. References 1. Mohanty M. Cellular basis for failure of joint prosthesis. Bio Med Mater Eng 1996;6:165-72. 2. Rockborn P, Olsson SS. Loosening and bone resorption in Exeter hip arthroplasties: review at a minimum of five years. J Bone Joint Surg [Br] 1993;75-B:865-8. 3. Santavirta S, Konttinen YT, Bergroth V, et al. Aggressive granulomatous lesions associated with hip arthroplasty: immunopathological studies. J Bone Joint Surg [Am] 1990;72-A:252-8. 4. Greitemann B, Mues B, Polster J, Pauly T, Sorg C. Inflammatory reactions in primary osteoarthritis of the hip and total hip prosthesis loosening. Arch Orthop Trauma Surg 1992;111:138-41. 5. Boynton EL, Henry M, Morton J, Waddell JP. The inflammatory response to particulate wear debris in total hip arthroplasty. Can J Surg 1995;38:507-15. 6. Jiranek WA, Machado M, Jasty M, et al. Production of cytokines around loosened cemented acetabular components: analysis with immunohistochemical techniques and in situ hybridization. J Bone Joint Surg [Am] 1993;75-A:863-79. 7. Kim KJ, Chiba J, Rubash HE. In vivo and in vitro analysis of membranes from hip prostheses inserted without cement. J Bone Joint Surg [Am] 1994;76-A:172-80. 8. Horikoshi M, Macaulay W, Booth RE, Crossett LS, Rubash HE. Comparison of interface membranes obtained from failed cemented and cementless hip and knee prostheses. Clin Orthop 1994; 309:69-87. 9. Chiba J, Rubash HE, Kim KJ, Iwaki Y. The characterization of cytokines in the interface tissue obtained from failed cementless total hip arthroplasty with and without femoral osteolysis. Clin Orthop 1994;300:304-12. 10. Shanbhag AS, Jacobs JJ, Black J, Galante JO, Glant TT. Cellular mediators secreted by interfacial membranes obtained at revision total hip arthroplasty. J Arthroplasty 1995;10:498-506. 11. Ziff M. Pathways of mononuclear cell infiltration in rheumatoid synovitis. Rheumatol Int 1989;9:97-103. 12. Gowen M, MacDonald BR, Hughes DE, Skjodt H, Russell RGG. Immune cells and bone resorption. Adv Exp Med Biol 1986;208: 261-73. 13. Bjurholm A, Kreicbergs A, Brodin E, Schultzberg M. Substance Pand CGRP-immunoreactive nerves in bone. Peptides 1988;9:165-71. 14. Bjurholm A, Kreicbergs A, Ahmed M, Schultzberg M. Noradrenergic and peptidergic nerves in the synovial membrane of the Sprague-Dawley rat. Arthritis Rheum 1990;33:859-65. 15. H¨okfelt T, Kellerth JO, Nilsson G, Pernow B. Substance P: localization in the central nervous system and in some primary sensory neurons. Science 1975;190:889-90. 16. Go VLW, Yaksh TL. Release of substance P from the cat spinal cord. J Physiol 1987;391:141-67.
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